The success of the reversible thiol–disulfide exchange method developed in this work is dependent on the premise that the tetrameric form of the peptide should form disulfides more readily than the monomeric form, thereby allowing one to mathematically extract the dissociation constant for tetramerization interaction. Conversely, if this is correct the disulfide-bonded dimer (DSS) should form disulfide-bonded tetramers (TSS,SS) at concentrations significantly lower than those required to form fully reduced tetramers (TSH,SH) from the corresponding reduced monomers (MSH) (Scheme 1). We used analytical ultracentrifugation to test this possibility.
The equilibrium association of M2TM19–46 peptide in both reduced and oxidized states was determined by sedimentation equilibrium in DPC micelles utilizing methods analogous to those used to study other transmembrane fragments of M2 (Kochendoerfer et al. 1999; Salom et al. 2000; Howard et al. 2002). The monomeric and dimeric (oxidized) peptides were incorporated into DPC micelles at a DPC concentration (15 mM) higher than its critical micelle concentration (1 mM; Kochendoerfer et al. 1999). To eliminate the contribution of the DPC micelles to the peptide's sedimentation, the density of the solvent was adjusted with D2O to match the density of the detergent (Tanford et al. 1974).
The M2TM19–46 peptide is essentially fully insoluble in water, in both the reduced and oxidized states. Thus, the appropriate concentration variable for analyzing the sedimentation equilibrium curves is the activity of the peptide in the micellar phase, which can be approximated as the mole fraction (MF) of the peptide (peptide/detergent ratio). The equilibrium radial concentration profile for the reduced peptide is well described by a fully cooperative monomer–tetramer equilibrium with a Kd of 2.0 × 10−7 MF3 for the tetrameric dissociation constant (see supplemental Fig. S1-curve a). This value is identical to that reported previously for 22–46 TM segment (Salom et al. 2000), and corresponds to a midpoint of 1:136 (peptide/detergent ratio). From the sedimentation data for the oxidized peptide (see supplemental Fig. S1-curve b), a dissociation constant of 6.2 × 10−5 MF was derived for the monomer–dimer dissociation constant, which corresponds to a midpoint of 1:16,000 (peptide/detergent ratio).
These two results indicate that the reduced and oxidized forms of M2TM19–46 exist in a reversible equilibrium in DPC micelles, and that the oxidation greatly stabilizes the tetrameric (TSS,SS) form of the protein.
Equilibrium studies of M2TM19–46in detergent micelles
The above sedimentation studies indicate that the redox behavior of the M2TM peptide should depend on the peptide/detergent ratio, the peptide becoming more easy to oxidize as the concentration of peptide is increased. To test this prediction, the reduced form of M2TM19–46 peptide was incorporated into DPC at various peptide-to-detergent molar ratios, in the presence of glutathione redox buffer containing known concentrations of oxidized (GSSG) and reduced (GSH) glutathione. The reaction was allowed to proceed to equilibrium, quenched by addition of HCl to effectively eliminate thiol exchange and oxidation, and the components present in the equilibrium mixture separated and quantified by analytical RP-HPLC.
Our first objective was to determine conditions under which the reaction was rapid and reversible. The formation or reduction of disulfide bonds by exchange with a linear or cyclic disulfide–thiol reagent requires formation of a mixed disulfide intermediate (Creighton and Goldenberg 1984). Reagents such as glutathione promote the formation of stable mixed disulfides with protein thiols (Konishi et al. 1981; Creighton and Goldenberg 1984; Creighton 1986; Lin and Kim 1989). Utilization of too high concentrations of glutathione can lead to formation of mixed disulfides in high amounts, thus limiting the formation of the disulfide species of interest. Therefore, to determine the optimum conditions for disulfide cross-linking, different conditions were tested. The equilibration reactions were carried out by varying both the GSSG/GSH ratio and the total concentration of the two components. The oxidized and reduced glutathione forms were used in large excess over the peptide such that their concentrations at equilibrium can be considered unchanged while analyzing the equilibrium. Figure 1 shows a typical set of HPLC chromatograms of different equilibrium mixtures in a glutathione redox buffer containing 10.6 mM total glutathione and different GSSG/GSH ratios. Three major peaks are observed corresponding to the reduced (MSH) and oxidized (MSSM) peptides and mixed monodisulfide derivative (MSSG) of the peptide with glutathione. As Figure 1 indicates, the oxidized peptide accumulates under increasingly oxidizing conditions. However, an appreciable amount of mixed disulfide intermediate species forms and becomes kinetically trapped. Therefore, to reduce the extent of mixed disulfide species formation, we carried out the cross-linking measurements at a lower concentration of glutathione (1.5 mM). Figure 2 compares the amount of dimer generated in the experiments conducted at 10.6 mM and 1.5 mM total glutathione for two different peptide-to-detergent mole ratios. It is evident from Figure 2 that the higher concentration of total glutathione leads to a greater tendency to form mixed disulfides between glutathione and protein, thus limiting the yield of dimer. By reducing the concentration of glutathione, the extent of dimer formation is significantly enhanced, and the process is readily reversible. When equilibration is carried out starting with the oxidized peptide (dimer) incorporated into DPC micelles, the two forms accumulate to the same extent as when the equilibrium is approached from the other direction by starting with the reduced peptide (see supplemental Fig. S2).
The sedimentation equilibrium study showed that the peptide exists in a reversible monomer–tetramer equilibrium, which depends on the amount of detergent, favoring monomers at low peptide-to-detergent ratios and tetramers at high peptide-to-detergent ratios. Therefore, one might anticipate that the extent of cross-linking be dependent on the peptide-to-detergent ratio; an enhanced ability to form disulfide-linked species is expected at high peptide-to-detergent ratios, which diminishes at low peptide-to-detergent ratios.
The percent of disulfide cross-linked product obtained from equilibration reactions carried out at different peptide-to-detergent mole ratios is plotted as a function of the GSSG/GSH ratio in Figure 3. Formation of oxidized species in Figure 3 is dependent on the peptide-to-detergent mole ratio in the expected qualitative manner. At high peptide-to-detergent mole ratios, the concentration of the protein in the micellar phase is increased and disulfide formation is enhanced, as reflected in the increase of the amount of dimer formed at equilibrium. At low peptide-to-detergent mole ratios the protein becomes predominantly monomeric leading to a decrease in the extent of cross-linking. Thus, the percentage of total protein that exists as a dimer at equilibrium is diminished.
To confirm that the protein specifically associates into tetramers, we conducted the disulfide cross-linking experiments in the presence of amantadine. Previous studies showed that 22–46 TM segment of M2 forms amantadine-sensitive ion channels. and that binding of the drug favors tetramer formation (Salom et al. 2000). To investigate the effect of amantadine on the oligomerization of M2TM19–46, we conducted the disulfide bond formation experiments in the presence of amantadine. For this study, the peptide was incorporated into DPC at a low peptide to detergent ratio (1:1000); well below the midpoint for tetramer formation by the monomeric peptide. Different amounts of amantadine were added to the peptide/DPC samples and disulfide formation was initiated by adding GSSG and GSH in varying ratios. Figure 4 shows that addition of amantadine results in an enhancement of the cross-linking. The shift in the monomer–tetramer equilibrium induced by amantadine indicates that the protein specifically associates into tetramers, which bind amantadine.
The model employed to describe the system and to account for various species present in the equilibration mixture is presented in Scheme 2.
K1 represents the tetrameric dissociation constant, K2 is the equilibrium constant for the formation of the first disulfide bond, K3 is the equilibrium constant for the second disulfide bond formation, whereas K4 represents the dissociation constant for the dimer–tetramer equilibrium. MSH is the reduced, monomeric peptide and TSH,SH is the reduced, tetramer. TSS,SH, TSS,SS, and DSS are intermediate species and represent the one-disulfide tetramer, fully oxidized tetramer, and the oxidized peptide (disulfide-bonded dimer), respectively. The values of K1 and K4 were determined in the above section on analytical ultracentrifugation. These values will be compared with those derived from fitted parameters in the model presented in Scheme 2. The only constants that depend on the redox potential of the solution, expressed as [GSSG]/[GSH]2, are K2, K3, and K6.
Because the redox buffer used in the above disulfide measurements contains very low starting GSSG to GSH ratios, that is, low concentrations of oxidized glutathione, quantitation of the data in the region corresponding to these low concentrations (Fig. 3) was prone to errors (because the equilibrium concentration of GSSG is low). Consequently, globally fitting the data in Figure 3 has proven to be problematic. To circumvent this problem, we conducted the equilibrations at sufficiently high GSSG to GSH ratios, where a more accurate measurement of the data is possible. The disulfide cross-linking reactions were carried out at three different fixed GSSG/GSH ratios (0.5, 0.2, and 0.1), and various peptide-to-detergent mole ratios.
Figure 5 illustrates the percentage of disulfide-bonded dimer generated from the three different experiments. The curves associated with the data in Figure 5 are fits obtained using the model in Scheme 1 (for details, see supplemental material). In fitting the data, we made the simplifying assumption that the intermediate state DSS is populated at very low levels. This supposition is supported by the sedimentation equilibrium data, which indicate (a) a cooperative monomer–tetramer equilibrium and (b) a tight dimer to tetramer association.
Under these conditions, the total thermodynamic equilibrium is described by K1 through K4, and it is not necessary to specify K5 and K6. Further, to test the effect of K5 and K6 on the fit and the parameters obtained, the data were also fit to a model including K5 and K6. No improvement in the fit was observed, nor were K1 and K4 affected, indicating that the most significant thermodynamic pathway is the one described by K1 through K4. The values of the logarithm of any one of these parameters could be varied by up to 10% without significantly affecting the quality of fit of the curve to the data.
On the basis of the good fits obtained in Figure 5, it appears that the model illustrated in Scheme 1 describes the system very well. In Table 1 the dissociation constants derived from fitting the cross-linking data are reported as pKdiss values, and are compared to those obtained from analytical ultracentrifugation. As Table 1 shows, the values obtained for pK1 and pK4 from the cross-linking measurements are within experimental error and in consonance with those estimated from the equilibrium sedimentation study. pK2 and pK3 values are also in good agreement for measurements at all three GSSG/GSH ratios with the exception of pK3 at the lowest GSSG/GSH ratio employed (0.1). The equilibrium constant for the first disulfide bond formation (K2) is in the range of 3–7 M, whereas formation of the second disulfide bond has an equilibrium constant (K3) of ∼1.5 M (for the latter, only the two K3 values corresponding to the highest GSSG/GSH ratios utilized (0.5 and 0.2) were considered—see Table 1). These constants are often considered as an effective concentration of the Cys residues, providing a quantitative measure of how readily each disulfide forms. The values obtained are very close to the range of values typically observed for Cys residues in native folded proteins (5–20 M, although values as high as 1 × 105 M have been measured) (Lin and Kim 1989; Regan et al. 1994). Thus, these values are consistent with the Cys residues being presented from expected locations within short partially loops, which project from the same side of the bundle. The data also indicates that the formation of the first disulfide bond within the tetramer is more favorable than that of the second disulfide bond. Based on statistical considerations, one might indeed expect that the second disulfide bond will form with an equilibrium constant, K3, of ∼(1/3) K2, where 3 is a statistical factor that accounts for the fact that a given Cys has three potential partners in the fully reduced tetramer, but only one in the half-oxidized structure. (A given Cys residue has two potential partners on neighboring helices and one partner on the diagonally opposed helix in the tetramer.)
The good agreement between pK1 and pK4 values and those obtained from the analytical ultracentrifugation study strongly indicates that disulfide cross-linking can be used as a quantitative method to measure the association in detergent micelles.